The invention is generally in the field of biosensors, and concerns a sensor useful for the determination of the presence, and optionally also concentration, of an analyte in a liquid, particularly aqueous, medium. The present invention relates to such electrodes, as well as their use and systems comprising them.
In the following description, reference will be made to several prior art documents shown in the list of references below. The reference will be made by indicating their number from this list.
References
1. E. Engvall, in: Methods in Enzymology, Vol. 70, 1980, pp. 419-439.
2. A. Shons, F. Dorman, J. Najarian, J. Biomed. Mater. Res. 6, 565 (1972).
3. A. A. Suleiman and G. G. Guilbault, Analyst, 119, 2279 (1994).
4. M. D. Ward and D. A. Buttry, Science, 249, 1000 (1990).
5. J. R. Oliveria and S. F. Silver, U.S. Pat. No. 4,242,096 (1980).
6. T. K. Rice, U.S. Pat. No. 4,236,893 (1980).
7. T. K. Rice, U.S. Pat. No. 4,314,821 (1982).
8. J. E. Roederer, G. J. Bastiaans, Anal. Chem., 55, 2333 (1983).
9. J. E. Roederer and G. J. Bastiaans, U.S. Pat. No. 4,735,906 (1988).
10. H. Muramatsu, J. M. Dicks, E. Tamiya and I. Karube, Anal. Chem., 59, 2760 (1987).
11. D. Mueller-Schulte and H. Laurs, CA. 1990, 112(7), 51807 g.
12. H. Muramatsu, K. Kajiwara, E. Tamiya and I. Karube, Anal. Chim. Acta, 188, 257 (1986).
13. H. Muramatsu, Y., Watanabe, M. Hikuma, T. Ataka, I. Kubo, E. Tamiya and I. Karube, Anal. Lett., 22, 2155 (1989).
14. B. Konig and M. Grxc3xa4tzel, Anal. Lett., 26, 1567 (193).
15. M D. Ward and R. C. Ebersole, PCT Application, Application No. WO 89/09937.
16. R. C. Ebersole, R. P. Foss and M. D. Ward, PCT Application, Application No. WO/94/02852.
17. R. C. Ebersole and J. R. Moran, PCT Application, Application No. WO/91/05251.
18. N. J. Geddes, E. M. Paschinger, D. N. Furlong, F. Caruso, C. L. Foffmann and J. F. Rabolt, Thin Solid Films, 260:192-199 (1995).
19. I. Willner, S. Rubin and Y. Cohen, J. Amer. Chem. Soc., 115:4937-4938, (1993).
20. I. Willner, R. Blonder and A. Dagan, J. Amer. Chem. Soc., 116:9365-9366, (1994).
Mention of the above references in this writing does not mean to imply that these references are in any way relevant to the issue of patentability of the invention as defined in the appended claims.
The specificity of antigen-antibody binding interactions and the technological progress in eliciting monoclonal antibodies for low molecular weight materials provide the grounds to design sensitive immunosensor devices for clinical diagnostics, food control and environmentally polluting substances. The most extensively developed immunosensor analyses include radioisotopic antigen/Ab labels and enzyme-linked immunosorbant assays (ELISA)(1).
The discovery of a linear relationship between the change in the oscillating frequency of a piezoelectric crystal and the mass variation on the crystal as a result of binding or adsorption phenomena opened the possibilities to monitor gravimetrically antigen-antibody binding phenomena. The mathematical relation between the frequency changes of a piezoelectric crystal, xcex94f, and mass changes, xcex94m, on the crystal is given by the following Sauerbrey equation:
xcex94f=xe2x88x922.3xc3x97106fo2xc2x7xcex94m/A 
where fo is the fundamental resonance frequency of the crystal prior to the mass variation and A is the surface area of deposited mass. For example, for a crystal exhibiting a fundamental frequency of 9 MHz and surface area of 1 cm2, a mass-change on the crystal that corresponds to 1xc3x9710xe2x88x929 g will stimulate a frequency change, xcex94f, of 6 Hz.
The first analytical use of piezoelectric crystals in relation to antigen-antibody (Agxe2x80x94Ab) interactions was reported in 1972(2), where a nyebar precoated crystal was further coated via hydrophobic interactions, with bovine serum albumin (BSA) and the association of the BSAxe2x80x94Ab to the crystal was monitored by the frequency changes. Since then, the piezoelectric detection of antigens and antibodies by piezoelectric means or the quartz crystal microbalance (QCM) has been adopted in a series of analytical studies. The progress in this area has been reviewed by Suleiman et al., 1994(3) and Ward et al., 1990(4). Immobilization of an antibody on a QCM device has been described by Geddes et al.(18).
Several patents describe the application of QCM for the analysis of antigens and antibodies. Physical adsorption of antigens to a crystal was used as a means for the detection of antigens by interacting the crystal with a mixture of the analyte antigen and a predetermined amount of Ab(5). The decrease in the antigen concentration was inversely related to the antigen concentration in the sample. In two patents by Rice(6,7), methods for the determination of Abs by QCM were disclosed. The antigen was immobilized on a polymer precoated crystal and the frequency changes as a result of Ab association related to the analyte Ab concentration in the sample. By this method, human IgG against honey bee venom, phospholipase A, and keyhole limpet hemocyanine were analyzed(6). However, non-specific binding to the crystal interfered with the analyses. In a follow-up patent(7), the detection of low molecular weight components by a pre-coated crystal with the anti-Ab and competitive binding assay of the Ab-low molecular weight analyte was described. All of these analyses were performed by treatment of the crystals in solution and subsequent frequency measurements in air. This two-step solution/gas procedure allows improvement of the sensitivity of the resonating QCM, but introduces technical complications and the interference of hydration/dehydration phenomena that are reflected in the frequency parameters. Ward et al. (15) and Ebersole et al. (17) disclose a QCM assay where the sensitivity is increased by the use of an enzyme comprising conjugates which binds to the analyte after the latter has been bound to a capturing agent, which enzyme catalyzes a reaction where a substrate is converted to the product and the product which is absorbed on the QCM increases the mass of the QCM which gives rise to a change in its resonance frequency. Ebersole et al.(16) discloses a method that makes use of a polymer which changes its mass in the presence of an analyte, e.g. H+ ions (serving as a pH) sensor.
Piezoelectric immunoassaying in the liquid phase has important technical advantages as it allows stationary and flow analysis of aqueous samples. The method suffers, however, from a basic physical limitation due to substantially lower frequency changes of the crystal as a result of the solution viscosity. QCM immunoassays in solution were reported by Roederer(8) and addressed in a follow-up patent(9). The quartz crystal was modified with glycidoxypropyltrimethoxy silane (GOPS), and the surface-modified crystal was then further modified by anti-human IgG antibody and then applied for the piezoelectric detection of human IgG. The detection limit of the device was determined to be 13 xcexcgxc2x7mlxe2x88x921. A closely related approach was adopted by Muramatsu et al.(10) where the quartz crystals were surface-modified by xcex3-aminopropyl triethoxy silane and further derivatized by protein A. The surface-modified crystals were then applied for the determination of human IgG in the concentration range 10xe2x88x926-10xe2x88x922 mgxc2x7mlxe2x88x921. A related patent disclosed the piezoelectric analysis of thyroxine using a polyamide 6 polymer coating and anti-thyroxine Ab as sensing interface(11).
Piezoelectric analysis of high molecular weight antigens such as microbial cells was addressed using antibody-coated quartz crystals. C. albicans cells in the concentration range 1xc3x97106-5xc3x97108 cellsxc2x7mlxe2x88x921 were analyzed by an anti-Candida albicans Ab surface (12), E. coli with an anti-E. coli interface(13) and protein A-coated crystals acted as piezoelectric sensing interface for various bacteria including Salmonella, Shigella, Yersinia and E. Coli(14).
Use of photoisomerizable substance for the photoregulated binding of molecules to a substrate has been described by Willner et al.(19) The aplication of this feature in reversible amperometric immunosensors has been described by Willner et al.(20) 
Methods utilizing piezoelectric devices allow immuno-chemical sensing of interactions between two members of a recognition pair such as Abxe2x80x94Ag, sugar-lectin, biotin-avidin, etc., without the need for labeling, and provide competitive analytical tools to conventional radio-labeled and enzyme-labeled analyses.
It is an object of the present invention to provide a method for determining the presence and optionally the concentration of analyte in a liquid medium, analyte being a member of a recognition pair.
It is further an object, in accordance with an embodiment of the present invention, to provide a system for carrying out the above method.
It is furthermore an object of the present invention to provide electrodes for use in the above system and method.
It is still further an object of the present invention to provide a process for the preparation of such electrodes.
The present invention makes use of a piezoelectric crystal and determining a change in mass bound to the crystal by measuring a change of its resonance frequency. In the following, the term xe2x80x9cxcex94f responsexe2x80x9d will be used to denote a change of frequency of the electrode as the result of binding of a mass thereto or release of a mass therefrom.
In accordance with the present invention, a novel system and an electrode for use in the system are provided. The system in accordance with the present invention is capable, by means of a xcex94f response, to determine the presence and optionally the concentration of an analyte in a liquid medium. The analyte is a member of a pair of molecules or complexes of molecules, which can specifically bind to one another in a non-covalent manner. Such a pair of molecules will be referred to herein as xe2x80x9crecognition pairxe2x80x9d. The recognition pair may consist for example of antigen-antibody, ligand-receptor, sugar-lectin, biotin-avidin, enzyme-substrate, oligonucleotide-oligonucleotide with a complementary sequence, oligonucleotide-protein, olignucleotide-cell, etc.
In the following description the terms xe2x80x9cdeterminationxe2x80x9d or xe2x80x9cdeterminexe2x80x9d will be used to denote both qualitative and quantitative determination of binding. Where, for example, the method and system defined below are used for determining an analyte in a liquid medium, this is meant to denote determining the presence of an analyte in the medium and optionally its concentration. In other words, a xcex94f response will be used as a qualitative measure for the presence of the analyte in a medium; the extent of the xcex94f response will be used as a measure of the amount of analyte in a tested medium.
The term xe2x80x9canalytexe2x80x9d already used above and which will be used further below, is meant to denote an unknown agent determined in a liquid medium.
The present invention has several aspects. One such aspect concerns a system for determining binding between two members of a recognition pair (xe2x80x9csystem aspectxe2x80x9d); another such aspect relates to a method for determining such binding, which may be used for testing an analyte in a medium (xe2x80x9cmethod aspectxe2x80x9d); a further aspect is concerned with probes for use in the above system and method (xe2x80x9cprobe aspectxe2x80x9d); and a further aspect is concerned with a process for the preparation of such a probe (xe2x80x9cprocess aspectxe2x80x9d).
In accordance with the system aspect of the present invention, there is provided a system for determining binding between two members of a recognition pair, comprising:
(a) a probe comprising a piezoelectric crystal, electrodes on two opposite faces of the crystal, and one or more metal plates carried on the surface of said crystal, said metal plates being the same or different than said electrodes, the metal plates having immobilized thereon a first member of a recognition pair, binding of a second member of the recognition pair to the first member, or dissociation between the two members and release of the second member from the probe, causing a change of mass resulting in a change to the probe""s resonance frequency;
(b) a vessel for holding a liquid, the probe being immersed in the liquid to allow either
binding between the first, immobilized member and the second member dissolved in the liquid, or
release of the second member, a priori bound to said first member, into said liquid; and
(c) electric or electronic circuitry for generating an alternating electric field between said electrodes, and measuring of the resonance frequency of said crystal.
In accordance with an embodiment of the method aspect of the invention, there is provided a method for determining binding between a first member of a recognition pair and a second member of a recognition pair, the second member being a priori contained in a liquid medium, comprising:
(a) providing a probe comprising a piezoelectric crystal, electrodes on two opposite faces of the crystal, and comprising one or more metal plates carried on the surface of said crystal, said plates being the same or different than said electrodes, the first member of the recognition pair being immobilized on the said plates;
(b) measuring an initial resonance frequency of the probe;
(c) contacting said probe with a liquid medium containing said second member for a time sufficient to allow binding between the two members; and
(d) measuring a second resonance frequency, a lower second resonance frequency as compared to the initial resonance frequency indicating the presence of said second member in the liquid medium.
In accordance with another embodiment of the method aspect, there is provided a method for determining an analyte in a liquid medium, comprising:
(a) providing a probe comprising a piezoelectric crystal, electrodes on two opposite faces of the probe, and comprising one or more metal plates carried on the surface of said crystal, said metal plates being the same or different than said electrodes, the metal plates having immobilized thereon a first member of a recognition pair, the second member of said pair being non-covalently bound to said first member, said second member being capable of binding to said analyte, the binding between said second member and said analyte being competitive to the binding of said second member to said immobilized member;
(b) measuring an initial resonance frequency of the probe;
(c) contacting said probe with said liquid medium under conditions and for a time such that in the presence of said analyte, at least some of said second member will be released from the electrode and bind to said analyte; and
(d) measuring a second resonance frequency, a higher second resonance frequency as compared to the initial resonance frequency indicating the presence of said analyte in said medium.
In accordance with a further embodiment of the method aspect, there is provided a method for determining an analyte in a liquid medium, comprising:
(a) providing a probe comprising a piezoelectric crystal, electrodes on two opposite faces of the crystal, and comprising one or more metal plates carried on the surface of said crystal, said metal plates being the same or different than said electrodes, said metal plates having immobilized thereon a first member of a recognition pair; the pair comprising a second member being capable of binding to said analyte, the binding between said second member and said analyte being competitive to the binding of said second member to said immobilized member;
(b) measuring an initial resonance frequency of the probe;
(c) mixing said liquid medium with a solution containing said second member, the presence of said analyte in the medium causing binding thereto of said second member;
(d) contacting the mixture obtained in step (c) with said probe for a time sufficient to allow binding of said second member to the immobilized first member; and
(e) measuring a second resonance frequency of the probe, a second resonance frequency lower than the initial frequency indicating pressence of said analyte in the liquid medium (a relatively large decrease in resonance frequency meaning no or a small amount of analyte in the liquid medium; no or a small decrease in resonance frequency meaning a relatively large amount of the analyte in the liquid medium).
In accordance with the probe aspect of the invention there is provided a probe for use in the above method and system. The probe comprises a piezoelectric crystal having electrodes on two opposite faces of the crystal, and one or more metal plates carried on the surface of said crystal, said metal plates being the same or different than said electrodes, the metal plates having immobilized thereon a first member of a recognition pair.
In order to cause a piezoelectric crystal to vibrate and eventually reach resonance frequency, the piezoelectric crystal has to be subjected to an alternating electrical field. The piezoelectric crystal used in accordance with the invention is typically a planar crystal having the form of a plate or a disc, and the electrodes which provide the alternating electrical field are typically planar metal electrodes attached to opposite faces of the crystal. The plates with the immobilized member of a recognition pair are preferably the same as the planar electrodes, in other words the electrodes serve both for the provision of an alternate electrical field and for immobilization of said first member.
An embodiment in accordance with the present invention where the analyte is measured directly by binding to the first member immobilized on the probe, will be referred to herein as xe2x80x9cdirect embodimentxe2x80x9d. A direct embodiment is an embodiment where the analyte to be determined is the second member of the recognition pair. An embodiment in accordance with the invention, such as the second and third embodiments defined above, where the presence of analyte is measured indirectly, i.e. what is measured in essence is the depletion of the second member will be referred to herein as the xe2x80x9cindirect embodimentxe2x80x9d.
The method in accordance with the direct embodiment can be practiced in particular where the second member is a relatively large molecule or a complex of molecules, the binding of which to the immobilized member causing a considerable mass change. Where the analyte is a small molecule, it is usually preferred to practice the invention by an indirect embodiment, since binding of such an analyte to the probe will bring about only a very small change of mass. The second member in such a case will typically be a large molecule, e.g. an antibody with a binding affinity to said analyte.
An example of the direct embodiment of the invention is the determination of an antibody in a biological sample in which case the electrode has immobilized thereon an antigen to which said antibody specifically binds; or the determination of a protein antigen by the use of an electrode having immobilized thereon an anti-antigen antibody.
In accordance with the indirect embodiment, the immobilized member may be an immobilized analyte molecule or a molecule with a similar binding specificity to said second member as said analyte. Preferably, the immobilized analyte molecule has a lower binding affinity to said second member than the analyte, to allow effective depletion of said second member in the presence of the analyte.
An example of the indirect embodiment is the use of an immobilized antigen in order to determine an identical or related antigen in a biological sample to be tested. In accordance with this specific example, the biological sample, e.g. a plasma sample is first reacted with a reagent solution comprising an antibody which specifically binds to the antigen to be determined. After binding, the concentration of free (unbound) antibody becomes lower. Following an incubation period, a probe having antigen molecules immobilized thereon (the immobilized antigen, in this case being said immobilized member) is challenged with the reacted solution, and the determination of the free antibody then serves as an indication of said antigen in the tested biological sample. As will no doubt be appreciated by the artisan, the concentration of said free antibody will be in opposite correlation to the concentration of the antigen in the tested sample.
Furthermore, as will also be appreciated, an antibody in a tested biological sample rather than an antigen may be determined in an analogous manner, mutatis mutandis.
The analyte may at times also be a molecule suspended or dissolved in a gas, e.g. various airborne chemicals. In such a case, a gas suspected of containing an analyte is first passed (e.g. xe2x80x9cbubbledxe2x80x9d) through a suitable liquid which can dissolve the analyte, and this liquid is then tested for the presence of the analyte therein. Obviously, as gaseous chemicals are typically small molecules, determining of such analyte is preferably carried out by the indirect embodiment.
At times, in order to increase sensitivity, rather than determining the xcex94f response within the liquid, the probe is first dried and then the measurement of xcex94f is performed with the probe embedded in a gas or in a vacuum.
The recognition pair, of which a first member is immobilized on the probe""s metal plate, may, for example, be an antigen-antibody, sugar-lectin, ligand-receptor, biotin-avidin, enzyme-substrate, oligonucleotide-complementary oligonucleotide, oligonucleotide-protein, and oligonucleotide-cell, and generally any pair of molecules with specific binding affinity to one another.
As a result of binding of the second member to the immobilized first member or the dissociation of the two members and the release of the second member from the probe, there is a change in mass which in turn results in a change in the resonance frequency (i.e. xcex94f response). The degree of xcex94f response correlates with the extent of binding or release of said second member and depends on the concentration of said analyte in the tested liquid surrounding the electrode. Thus, the extent of change in the resonance frequencies may be used, in accordance with a preferred embodiment of the invention, as an indication of the concentration of said analyte in the medium.
The metal plates carrying said immobilized member may be selected from a variety of metals, particularly such having the capability to associate chemically with, attach or chemisorb a sulphur-containing moiety. The metal plates are preferably made of or coated by metals such as gold, platinum, silver or copper.
The immobilized member is preferably immobilized on the surface of the metal plate by means of a linking group, which typically may have the following general formula (I):
Zxe2x80x94R1xe2x80x94Qxe2x80x83xe2x80x83(I) 
wherein:
Z represents a sulphur-containing moiety which is capable of chemical association with, attachment to or chemisorption onto said metal;
R1 represents a connecting group;
Q is a functional group which is capable of forming a covalent bond with a moiety of said first member of the recognition pair.
Z may for example be a sulphur atom, obtained from a thiol group, a disulphide group, a sulphonate group or sulphate groups.
R1 may be a covalent bond or may be a peptide or polypeptide or may be selected from a very wide variety of suitable groups such as alkylene, alkenylene, alkynylene phenyl containing chains, and many others.
Particular examples of R1 are a chemical bond or a group having the following formulae (IIa), (IIb), (IIc) or (IId): 
wherein
R2 or R3 may be the same or different and represent straight or branch alkylene, alkenylene, alkynylene having 1-16 carbon atoms or represent a covalent bond,
A and B may be the same or different and represent O or S,
Ph is a phenyl group which is optionally substituted, e.g. by one or more members selected from the group consisting of SO3xe2x88x92 or alkyl groups.
Q may for example be a functional group capable of binding to a carboxyl residue of a member of a recognition pair such as an amine group, a carboxyl group capable of binding to amine residues of the member of a recognition pair; an isocyanate or isothiocyanate croup or an acyl group capable of binding to an amine residue of the member of a recognition pair, or a halide group capable of binding to hydroxy residues of the protein or a polypeptide. Particular examples are the groups xe2x80x94NH2xe2x80x94COOH; xe2x80x94Nxe2x95x90Cxe2x95x90S; Nxe2x95x90Cxe2x95x90O; or an acyl group having the formulaxe2x80x94Raxe2x80x94COxe2x80x94G wherein G is a halogen such as Cl or OH, ORb, a 
group or a 
group; Ra and Rb being, independently a C1-C12 alkenyl, alkenyl or a phenyl containing chain which is optionally substituted, e.g. by halogen.
Particular examples of such a linking group are cysteamine (III), cystamine (IV) and cysteic acid N-hydroxysuccinimide ester (V) having the formulae: 
wherein n and m are integers between 1-24, preferably 1-12 and most preferably 1-6.
The sensitivity of the method of the invention may be increased by the use of a molecule, moiety or a complex, which is complexed or bound to said second member. Such a sensitivity increasing moiety, molecule or complex will be referred to herein as xe2x80x9camplifier groupxe2x80x9d. The amplifier group may be a molecule or a complex having a binding affinity to said second member. Such an amplifier group may be made to bind to said second member after same has bound to the immobilized member or prior to such binding. The binding or complexing of the amplifier group to said second member will increase the mass change as a result of binding of said second member, or dissociation and release of said second member, as the case may be, and accordingly there will be a more noticeable xcex94f response, and hence an increase in sensitivity.
By increasing the sensitivity of the system in the manner described above, a xcex94f response can be measured even after binding or release of only a few second member molecules to the probe.
Binding of two members of a binding couple to one another is typically a high affinity binding, namely the two members do not dissociate easily from one another and even after proper rinsing, the second member may still remain substantially bound to the first immobilized member. In order to re-use the probe for a further measurement, there is a need to dissociate the second member from the immobilized member and remove it from the system. In accordance with an embodiment of the invention, the dissociation is achieved by means of a group, attached to the immobilized member which has two isomerization states and is capable of switching reversibly between its two states by exposure to two different types of energy (xe2x80x9cisomerizable groupxe2x80x9d). Such an isomerizable group will typically have a first and second isomerization state and by reversibly switching from one state to the other, each such switching achieved with a different energy type, will cause a conformational change in the immobilized member which will bring about a change in the binding of affinity of the immobilized member to said analyte. Such a conformational change may, for example, be the occlusion of the binding site or a conformational change within the binding site which will cause a reduction in the binding affinity of the immobilized member to the second member. Such a reduction in affinity or vice versa may be defined as change or switch from a state of high affinity to a state of low, affinity or vice versa. In the first state, the immobilized member will have a high affinity to binding to the second member and after performing a measurement, the probe will be treated so that said isomerizable group will switch to the second state and consequently said second member will dissociate from the immobilized member. After removal of said analyte from the system, typically by rinsing and washing away of the rinsing solution, the probe will be further treated so that said isomerizable group switches back to said first state, whereby the probe will be ready for re-use.
The switching between the two states may be achieved by exposure to light of an appropriate wavelength within the infra red, visible or ultra violet range. The reactive isomerizable group will switch from said first state to said second state by exposure to light energy at a first wavelength and from a second state to said first state by exposure to a second, different than the first, wavelength. It is also possible that one of the switches will be achieved by mild thermal treatment.
Thus, in accordance with an embodiment of the invention the immobilized member of the recognition pair has or is linked to an isomerizable group reactive to exposure to light energy; said group having a first and a second state and is capable of being converted from the first state to the second state by exposure to irradiation of light of a first wavelength and from the second to the first state by exposure to irradiation of light of a second wavelength; the exposure inducing a change in affinity of the immobilized member for binding to said second member, whereby in the first state said immobilized member has a high affinity of binding to said second member such that said second member remains essentially bound to the immobilized member and in said second state said immobilized member has a low affinity of binding to said second member, such that the bound said second member is readily dissociated.
According to another embodiment of the invention said switching from the first state to the second state is by exposure to light energy but the switching from said second state to said first state is by mild thermal treatment.
In accordance with the process aspect of the invention, there is provided a process for preparing a probe for use in the above method and system, comprising:
(a) immobilizing said linking group onto the plate by chemical association attachment or chemisorption of the sulphur-containing moiety (Z); and
(b) binding the member of the recognition pair to be immobilized to said functional group (Q).
Steps (a) and (b) may also be reversed so that immobilization takes place before binding.
The process aspect of the invention further provides a process for preparing a probe carrying immobilized members which are attached to an isomerizable group, the process comprising:
(a) immobilizing said linking group onto the said plate by chemical association attachment or chemisorption of the sulphur-containing moiety;
(b) chemically modifying a member of said recognition pair with a photoisomerizable group whereby the modified member changes its binding affinity to the other member of the recognition pair by exposure to energy; and
(c) binding the modified member of the recognition pair to said functional group of the linking group immobilized on the electrode.
Steps (b) and (c) can be reversed such that the isomerizable group is bound to the member of the recognition pair after it has been immobilized in the electrode and so can steps (a) and (b).
The invention will now, be illustrated in the following description of some specific embodiments, with occasional reference to the annexed drawings, without prejudice to the generality of the foregoing.
In the drawings:
FIG. 1 shows a scheme of QCM for antibody analysis;
FIG. 2 shows a scheme for QCM analysis of an antigen by a QCM probe modified with an antigen monolayer and saturated with the Ab;
FIG. 3 shows a scheme for QCM analysis of an antigen by treatment of an antigen monolayer QCM probe with a mixture consisting of the analyte antigen and a constant, predetermined Ab concentration;
FIG. 4 shows a scheme for amplified QCM analysis of an Ab by the application of an anti-Ab or anti-Ab conjugate;
FIG. 5 shows an amplification of an antigen QCM analysis by the detachment of an Ab-conjugate complex from the QCM monolayer electrode;
FIG. 6 shows a possible configuration of Ab-conjugate complexes;
FIG. 7 shows an amplification of antigen QCM-analysis by treatment of an antigen monolayer QCM-probe with a mixture consisting of the antigen analyte and a fixed, predetermined, concentration of the Ab-conjugate complex;
FIG. 8 shows the regeneration of the sensing member by light isomerization;
FIG. 9 shows formulae of photoisomerizable groups and some examples of photo-induced and heat treatment-induced conformational changes;
FIG. 10 shows the organization of cystamine monolayer on a QCM gold (Au) electrode;
FIG. 11 shows a QCM-analysis of cystamine monolayer formation;
FIG. 12 shows the activation of the QCM-monolayer electrode by glutardialdehyde;
FIG. 13 shows QCM-analysis of the glutardialdehyde monolayer formation;
FIG. 14 shows the organization of HIV-1 antigen peptide on QCM Au-electrode:
FIG. 15 shows QCM-analysis of a 3000-titer serum of HIV-1 Ab;
FIG. 16 shows QCM-analysis of goat serum (titer 80) by the HIV-1 antigen electrode;
FIG. 17 shows the organization of a dinitrophenyl monolayer QCM electrode;
FIG. 18 shows a QCM-analysis of anti-DNP-Ab by dinitrophenyl antigen monolayer QCM electrode;
FIG. 19 shows organization of a fluorescein antigen monolayer QCM-electrode;
FIG. 20 shows QCM-analysis of 2,4-dinitrophenol, 1.4xc3x9710xe2x88x927 gxc2x7mlxe2x88x921, by detachment of DNP-Ab from the antigen-DNP-Ab monolayer electrode;
FIG. 21 shows the assembly of the dinitrophenyl antigen-DNP-biotin-avidin conjugate complex on a QCM-electrode;
FIG. 22 shows a scheme of QCM analysis of a sample solution that contains 2,4-dinitrophenol (DNP) by displacement of the dinitrophenyl antigen-DNP-biotin-avidin conjugate complex associated with a QCM electrode; and
FIG. 23 shows the xcex94f response in the system of FIG. 22 following exposure to 2.7xc3x9710xe2x88x928 gxc2x7mlxe2x88x921 of DNP.